This project is designed to determine the role of leptin receptor expression in neurons and adipocytes to the control of energy balance. The lack of leptin receptor (Lepr) results in a syndrome of obesity and diabetes. Investigations with leptin in humans and rodents suggest that the major weight-regulating action of leptin occurs in the hypothalamus. Specific neuronal populations, defined by their neuropeptide content such as NPY/AGRP and POMC/CART, are important mediators of leptin action. However, leptin has actions on adipocytes, such as the regulation of lipolysis, that may contribute to the obese phenotype. The goals of this proposal are: A) Evaluate the obesity-reducing effects of the expressing LeprB in neurons only, in adipocytes only, and in neurons and adipocytes; B) Evaluate the obesity-producing effects of abrogating leptin receptor expression in neurons only, in adipocytes only, and in neurons and adipocytes; C) Determine the obesity- and diabetes-reducing effects of expressing LeprB in POMC/CART neurons, in AGRP/NPY neurons, and in POMC/CART neurons and AGRP/NPY neurons. For A, transgenes with cell type-specific promoter/enhancers will be used to drive LeprB expression. The transgenes will be combined with the db-3J mutation, a 17bp deletion of coding exon 12 that prevents the synthesis of all membrane-bound LEPR isoforms. For B, the cre-lox system will be used to obtain tissue-specific lot deletion of coding exon 17 of Lepr. Homologous recombination in embryonal stem cells will be used to introduce two loxP sites that flank coding exon 17. The manipulated ES cells containing the floxed Lepr allele will be used to generate mice that carry Lepr-flox. Transgenes that express cre recombinase in a tissue- specific manner, neurons only or adipocytes only, will be bred with Lepr flox mice to obtain animals with tissue-specific deletion of Lepr expression. A gene knock-in method (C) will be used to identify specific neuronal populations that mediate the effects of leptin on obesity and diabetes. Expression of LeprB in chemically defined neurons will be achieved by the insertion of an IRES- LeprB/IRES-beta-Galactosidase cDNA cassette (internal ribosomal entry site) in the 3' untranslated regions of the Pomc and Agrp genes, generating a tricistronic mRNA. Thus, the effect of expression of LeprB in AGRP/NPY neurons and/or POMC/CART neurons on the obese/diabetic phenotype can be evaluated in db-3J/db-3J mice. These experiments will provide data regarding the role of leptin receptor signaling in AGRP/NPY neurons and POMC/CART neurons to the obesity/diabetes syndrome.